The complete herpes simplex virus type 2 envelope glycoprotein G was represented by overlapping synthetic peptides. Herpes simplex virus type 2-specific human seroreactivities were mainly seen against three peptides, peptides G2-64, G2-69, and G2-70, located in the C-terminal part of glycoprotein G. This is, interestingly, a region which has strong homology between herpes simplex virus types 1 and 2. G2-69 was the most herpes simplex virus type 2-specific peptide, reacting with 93% (13 of 14) of herpes simplex virus type 2 immunoglobulin G-positive human serum samples (n = 14) and none of the type 2 negative serum samples (n = 16) tested. The epitope of peptide G2-69 was mapped to the amino acid sequence RYAHPS, and the antibody binding to G2-64 could be increased by chemical oxidation of the peptide. The three reactive peptides were used, alone or combined, together with herpes simplex virus type 2 antigen in an attempt to discriminate anti-herpes simplex virus type 2 from anti-herpes simplex virus type 1 immunoglobulin G. Inclusion of the herpes simplex virus type 2 glycoprotein G peptides increased the specificity compared with the use of glycoprotein G alone. Combinations of peptides with whole glycoprotein also showed a greater discriminative capacity than single peptides. We conclude that these synthetic glycoprotein G peptides may be useful for herpes simplex virus type 2 serology based on peptides or combinations of peptides and antigens. This is the first study describing the possibility of discriminative herpes simplex virus serology by using synthetic peptides combined with small amounts of whole glycoprotein G.